Coxiella burnetii is an intracellular bacterial pathogen and the etiologic agent of human Q fever, an acute debilitating flu-like illness that can progress to chronic endocarditis. Since its discovery over 70 years ago, mechanisms used by the pathogen to parasitize host cells remain poorly understood. During infection, C. burnetii actively regulates multiple host processes, including vesicular trafficking and cell survival. The bacterial proteins mediating these events are not known but are likely delivered to the host cytosol by a Dot/Icm type IV secretion system. The current application is designed to functionally characterize C. burnetii plasmid-encoded Dot/Icm substrates and define their role in virulence. All C. burnetii isolates either harbor a large cryptic plasmid or have plasmid sequences integrated into their chromosome, suggesting these molecules are critical for pathogen biology. Interestingly, we have identified six Dot/Icm substrates encoded by C. burnetii plasmid genes that are termed Coxiella plasmid effectors A - F (CpeA - F). Three of these proteins are conserved in all isolates and three are specific to the QpH1 plasmid from a human acute disease isolate. Aim 1 is designed to characterize the interaction of conserved CpeB and CpeD with autophagosomes and secretory organelles, respectively. Aim 2 will define requirements of all six plasmid effectors during infection. Additionally, this aim will identify effector binding host proteins and determine the requirement of these components for C. burnetii infection. Aim 3 will determine the requirement of the C. burnetii plasmid for pathogen virulence in both cell culture and a guinea pig infection model of Q fever. Collectively, the aims in the current application will provide needed insight into the mechanisms used by C. burnetii to efficiently parasitize host cells. These studies will also provide novel information regarding the role of the C. burnetii cryptic plasmid in pathogen virulence. PUBLIC HEALTH RELEVANCE: Coxiella burnetii is an intracellular bacterial pathogen that causes the zoonosis human Q fever, a debilitating acute disease that also presents as chronic endocarditis. Currently, C. burnetii virulence determinants are poorly understood. Characterization of C. burnetii proteins delivered to the host cytosol by the Dot/Icm type IV secretion system will provide candidate therapeutic targets to combat Q fever and will provide needed insight into the interactions between C. burnetii and its host. The goals of the proposed research are to 1) characterize the interaction of C. burnetii plasmid-encoded Dot/Icm substrates with host proteins and 2) determine the role of the plasmid in pathogen virulence.)